脱氧雪腐镰刀菌烯醇（DON）人工抗原合成及其ELISA检测方法的研究
摘要
脱氧雪腐镰刀菌烯醇(Deoxynivalenol,DON)是禾谷镰刀菌的一种次级代谢产物,属单端孢霉烯族化合物,主要污染谷物及其制品,温带地区DON的污染比较严重,尤其是在开花季节或收割季节雨量偏多时。人和动物食用污染DON的粮食和饲料后不仅可导致急慢性中毒,而且与很多疾病密切相关。DON分布范围十分广泛,由于其对人和动物的危害及带来的经济损失,各国对DON的研究逐步深入,但是我国的相关研究相对较少,特别是检测水平偏低。因此,建立针对脱氧雪腐镰刀菌烯醇的快速检测方法在防止动物饲料真菌毒素中毒、控制食品的卫生质量、保障消费者健康、保证畜牧业正常发展等方面具有十分重要的社会意义和经济意义。
本论文通过对DON人工抗原合成的研究,建立了DON的酶联免疫吸附检测方法。用两种合成方法对脱氧雪腐镰刀菌烯醇与载体蛋白进行偶联,分别做为免疫抗原和包被抗原用于ELISA检测方法的研究,同时对ELISA检测条件进行优化,最终建立DON的ELISA检测方法,并对收集的样品进行检测分析,其主要研究内容和结果如下:
1、通过两种方法——琥珀酸酐法和N,N′-羰基二咪唑法实现了脱氧雪腐镰刀菌烯醇和大分子蛋白质(BSA/OVA)的偶联,并用紫外分光光度法和SDS-PAGE法对偶联物进行了定性检测,然后用ELISA试剂盒测得偶联物中DON的含量,其中与BSA偶联的偶联物中DON浓度为0.797μg/μL,与OVA偶联的偶联物中DON的浓度为0.518μg/μL。
2、将合成的DON人工抗原与等量佐剂混合后,采用颈背部皮下多点注射免疫昆明小鼠和Balb/c小鼠,每次免疫后采血,分离血清,并用ELISA检测方法检测血清效价,监测抗体产生情况,最终发现6次免疫后抗体效价最高。
3、对间接ELISA检测条件进行优化,包括包被液的包被时间和温度、一抗孵育时间、HRP酶标羊抗鼠抗体的孵育时间及底物加样后温育时间,并用L_(16)(4~5)正交表对这4个因素进行正交实验。正交实验证明,加好包被液的酶标板4℃冰箱过夜包被;一抗37℃温育时间是60 min;二抗37℃温育时间是60 min;加底物显色10 min后产生的阴阳性抗血清的OD_(450)差值最大。包被液的包被条件对实验结果的影响最大,其次是一抗和底物作用时间。由于二抗的温育时间对实验的结果影响不大,而且二抗温育30 min和温育120 min产生的实验结果相差很小。但从节约时间和不降低实验灵敏度的角度考虑,二抗的孵育时间定为60 min。
4、实验表明:乙腈-水(84:16)对DON具有更好的提取效果;加标回收率为86.4～95.9%;阳性临界值2.4;重复性和特异性良好。
5、间接竞争ELISA标准曲线方程是:Y=-37.945X+134.45,该法最低检出限为1μg/mL,线性范围为1～1000μg/mL,能达到饲料和畜产品中脱氧雪腐镰刀菌烯醇残留的检测要求。
6、用建立的ELISA检测方法对收集的各种样品进行检测,并与进口的ELISA检测试剂盒进行比较,差异不显著,可以用于样品脱氧雪腐镰刀菌烯醇的检测。
Deoxynivalenol(DON),the secondary metabolite of Fusatium fungi,is most member of the trichothecene family of mycotoxins.DON appears predominantly in wheat,corn,rye, rice and barley all over the world.The eatent of infection is dependent on weather conditions and storage conditions of cereal crops.Although DON is,aming the least toxic of the trichothecenes,it is the most frequently detected one throughout the word and its occurrence is considered to be an indicator of possible presence of other more toxic trichothecenes.In addition,DON is a very stable compound,during both storage and the processing/cooking of food,and it needs quite harsh conditions to get substantial breakdown.Consumption of contaminated feeds by livestock has been associated with a variety of adverse health effects including feed refusal,reduced weight gain,diarrhea and emesis.IARC had classified DON in group 3 "not classifiable as to its carcinogenicity to humand".
In the present study,we had produced deoxynivalenol antibody and had established DON's immunoassay method.We prepared immunogen of DON by two methods.Then dectected anti-sera titer from mice immunized with different immunogen.We optimized and established ELISA process.The main results were as following:
1.Synthesis immunogen with two methods—One was that DON obtained a carboxyl after deriving,and then complete antigen of DON was synthesized by succinic anhydride; The other was that DON was activated by 1,1-Diclohexylcarbodiimide.The products were detected by UV spectrum,SDS-PAGE and ELISA kit,and the concentration of DON in DON-BSAwas 0.797μg/μL,the DON-OVA was 0.518μg/μL.
2.Synthetical antigen was mixed with the same quantity adjuvant and immunized mice.After each immunization,the mice were blooded from tail.Separated sera,detected sera antibody titer by ELISA to monitor the variation of antibody.At last we conclude that the best antibody titer could be get after the sixth immunization.
3.The optimal working concentration of the coated antigen and the Balb/c mice sera dilutions in the indirect ELISA were 3μg/mL and 1:200,respectively.
4.Optimized indirect ELISA process.The best reaction-conditions were that plate coated at 4℃for 12 h,anti-sera incubated 60 min,IgG-HRP incubated 60min,substrate action time were 10min.
5.The experimentation showed:Samples were extracted with acetronile-water(84:16, V/V);recovery rate were 86.4～95.6%;cutoff value was 2.4;reproducibility and specificity were good.
6.The method of the indirect competitive ELISA for DON was established. Competitive inhibitory indirect ELISA standard curve equation was:Y=-37.945X+134.45. The detection limit was 1μg/mL.Samples with DON levels from 1 to 1000μg/mL could be quantitated.
7.Compared the ELISA with ELISA kit by detection of DON in samples,the result Show that no significant difference.
本论文通过对DON人工抗原合成的研究,建立了DON的酶联免疫吸附检测方法。用两种合成方法对脱氧雪腐镰刀菌烯醇与载体蛋白进行偶联,分别做为免疫抗原和包被抗原用于ELISA检测方法的研究,同时对ELISA检测条件进行优化,最终建立DON的ELISA检测方法,并对收集的样品进行检测分析,其主要研究内容和结果如下:
1、通过两种方法——琥珀酸酐法和N,N′-羰基二咪唑法实现了脱氧雪腐镰刀菌烯醇和大分子蛋白质(BSA/OVA)的偶联,并用紫外分光光度法和SDS-PAGE法对偶联物进行了定性检测,然后用ELISA试剂盒测得偶联物中DON的含量,其中与BSA偶联的偶联物中DON浓度为0.797μg/μL,与OVA偶联的偶联物中DON的浓度为0.518μg/μL。
2、将合成的DON人工抗原与等量佐剂混合后,采用颈背部皮下多点注射免疫昆明小鼠和Balb/c小鼠,每次免疫后采血,分离血清,并用ELISA检测方法检测血清效价,监测抗体产生情况,最终发现6次免疫后抗体效价最高。
3、对间接ELISA检测条件进行优化,包括包被液的包被时间和温度、一抗孵育时间、HRP酶标羊抗鼠抗体的孵育时间及底物加样后温育时间,并用L_(16)(4~5)正交表对这4个因素进行正交实验。正交实验证明,加好包被液的酶标板4℃冰箱过夜包被;一抗37℃温育时间是60 min;二抗37℃温育时间是60 min;加底物显色10 min后产生的阴阳性抗血清的OD_(450)差值最大。包被液的包被条件对实验结果的影响最大,其次是一抗和底物作用时间。由于二抗的温育时间对实验的结果影响不大,而且二抗温育30 min和温育120 min产生的实验结果相差很小。但从节约时间和不降低实验灵敏度的角度考虑,二抗的孵育时间定为60 min。
4、实验表明:乙腈-水(84:16)对DON具有更好的提取效果;加标回收率为86.4～95.9%;阳性临界值2.4;重复性和特异性良好。
5、间接竞争ELISA标准曲线方程是:Y=-37.945X+134.45,该法最低检出限为1μg/mL,线性范围为1～1000μg/mL,能达到饲料和畜产品中脱氧雪腐镰刀菌烯醇残留的检测要求。
6、用建立的ELISA检测方法对收集的各种样品进行检测,并与进口的ELISA检测试剂盒进行比较,差异不显著,可以用于样品脱氧雪腐镰刀菌烯醇的检测。
Deoxynivalenol(DON),the secondary metabolite of Fusatium fungi,is most member of the trichothecene family of mycotoxins.DON appears predominantly in wheat,corn,rye, rice and barley all over the world.The eatent of infection is dependent on weather conditions and storage conditions of cereal crops.Although DON is,aming the least toxic of the trichothecenes,it is the most frequently detected one throughout the word and its occurrence is considered to be an indicator of possible presence of other more toxic trichothecenes.In addition,DON is a very stable compound,during both storage and the processing/cooking of food,and it needs quite harsh conditions to get substantial breakdown.Consumption of contaminated feeds by livestock has been associated with a variety of adverse health effects including feed refusal,reduced weight gain,diarrhea and emesis.IARC had classified DON in group 3 "not classifiable as to its carcinogenicity to humand".
In the present study,we had produced deoxynivalenol antibody and had established DON's immunoassay method.We prepared immunogen of DON by two methods.Then dectected anti-sera titer from mice immunized with different immunogen.We optimized and established ELISA process.The main results were as following:
1.Synthesis immunogen with two methods—One was that DON obtained a carboxyl after deriving,and then complete antigen of DON was synthesized by succinic anhydride; The other was that DON was activated by 1,1-Diclohexylcarbodiimide.The products were detected by UV spectrum,SDS-PAGE and ELISA kit,and the concentration of DON in DON-BSAwas 0.797μg/μL,the DON-OVA was 0.518μg/μL.
2.Synthetical antigen was mixed with the same quantity adjuvant and immunized mice.After each immunization,the mice were blooded from tail.Separated sera,detected sera antibody titer by ELISA to monitor the variation of antibody.At last we conclude that the best antibody titer could be get after the sixth immunization.
3.The optimal working concentration of the coated antigen and the Balb/c mice sera dilutions in the indirect ELISA were 3μg/mL and 1:200,respectively.
4.Optimized indirect ELISA process.The best reaction-conditions were that plate coated at 4℃for 12 h,anti-sera incubated 60 min,IgG-HRP incubated 60min,substrate action time were 10min.
5.The experimentation showed:Samples were extracted with acetronile-water(84:16, V/V);recovery rate were 86.4～95.6%;cutoff value was 2.4;reproducibility and specificity were good.
6.The method of the indirect competitive ELISA for DON was established. Competitive inhibitory indirect ELISA standard curve equation was:Y=-37.945X+134.45. The detection limit was 1μg/mL.Samples with DON levels from 1 to 1000μg/mL could be quantitated.
7.Compared the ELISA with ELISA kit by detection of DON in samples,the result Show that no significant difference.
引文
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